Small-molecule inhibitors of the androgen receptor

ABSTRACT

The present invention provides tetrahydropyrvinium (THP), derivatives thereof, benzoxazole compounds, and derivatives thereof. The present invention provides a method of using tetrahydropyrvinium (THP), derivatives thereof, benzoxazole compounds, and derivatives thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. Ser. No.12/989,389, filed May 13, 2011, which entered the U.S. under 35 U.S.C.§371 from PCT/US2009/041715, filed Apr. 24, 2009, which claims priorityto U.S. Ser. No. 61/047,559, filed Apr. 24, 2008, herein incorporated byreference in their entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

Not applicable.

REFERENCE TO A SEQUENCE LISTING, A TABLE, OR A COMPUTER PROGRAM LISTINGAPPENDIX SUBMITTED ON A COMPACT DISK

Not applicable.

BACKGROUND OF THE INVENTION

Prostate cancer (PCa) is a leading cause of cancer morbidity andmortality in men, and the androgen receptor (AR) is the primarytherapeutic target. In early PCa, anti-androgen therapy (AAT) is almostuniversally effective. This consists of one or more combinations of GnRHagonists (to suppress pituitary signaling), aromatase inhibitors (todecrease androgen production), and competitive AR antagonists (to blockAR directly) such as hydroxy-flutamide (OH—F) or bicalutamide (BiC).This strategy usually works for several years, but over time tumor cellsevolve mechanisms for continued growth under these conditions ofandrogen depletion. Most recurrent, or hormone-refractory prostatecancer (HRPC) is nonetheless dependent on AR-mediated signaling. Thiscan include upregulation of AR protein expression levels, acquisition ofmutations within AR that increase its activity in response toalternative hormones (including antagonists), or upregulation ofco-activator proteins that augment AR activity. Thus, it is likely thatnew approaches to block AR activity could significantly extend orincrease the effectiveness of AAT. This could consist of bettercompetitive antagonists, and considerable efforts from pharmaceuticalcompanies are already being brought to bear on this approach. Thisimplies that novel anti-androgens might have considerable utility in thetreatment of both primary and recurrent PCa. Such anti-androgens mightnot be competitive antagonists that directly bind AR, and couldconceivably function via inhibition of downstream events in ARsignaling. Accordingly, there is a need for novel, potentanti-androgens. Surprisingly, this invention meets this, and other,needs.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the present invention provides compounds of FormulaI:

wherein each R¹ is independently hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl,C₂₋₆ alkynyl, C₁₋₆ alkoxy, —OR⁴, —SR⁴, —NR⁴R⁵, cycloalkyl,heterocycloalkyl, aryl or heteroaryl. R² is hydrogen, C₁₋₆ alkyl, C₁₋₆alkyl-OH, C₂₋₆ alkenyl or C₂₋₆ alkynyl. R³ is cycloalkyl,heterocycloalkyl, aryl or heteroaryl, optionally substituted with from 1to 3 R⁶ groups. R⁴ and R⁵ are each independently hydrogen, C₁₋₆ alkyl,C₂₋₆ alkenyl or C₂₋₆ alkynyl. Alternatively R⁴ and R⁵ are combined withthe nitrogen to which they are attached to form a heterocyclic ringhaving from 5 to 7 ring members and from 1 to 3 heteroatoms eachindependently N, O or S. Each R⁶ is independently H, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl or C₁₋₆ alkoxy. L is a linker of C₁₋₆ alkylene,C₂₋₆ alkenylene, C₂₋₆ alkynylene or C₃₋₆ cycloalkylene. X is —N(R⁷)—, anaryl ring having 6-10 ring members and a heteroaryl ring having from 5to 6 ring members and from 1 to 3 heteroatoms each independently N, O orS, wherein the aryl and the heteroaryl ring are each optionallysubstituted with from 1 to 3 R⁸ groups. R⁷ is H, C₁₋₆ alkyl, C₂₋₆alkenyl or C₂₋₆ alkynyl. Each R⁸ is independently H, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl or C₁₋₆ alkoxy. The compounds of Formula I includethe salts, hydrates and prodrugs thereof.

In a second embodiment, the present invention provides compounds ofFormula II:

wherein R¹, R³, R⁴, R⁵, R⁶, R⁷, R⁸, X and L are as defined above; R^(2′)is an electron pair, hydrogen, C₁₋₆ alkyl, C₁₋₆ alkyl-OH, C₂₋₆ alkenylor C₂₋₆ alkynyl; and Y is O or S. The compounds of Formula II includethe salts, hydrates and prodrugs thereof.

In a third embodiment, the present invention provides a pharmaceuticalcomposition including a compound of Formula I and a pharmaceuticallyacceptable excipient.

In a fourth embodiment, the present invention provides a method ofinhibiting an androgen receptor by administering to a patient in need ofsuch treatment, a therapeutically effective amount of a compound ofFormula I or Formula II.

In a fifth embodiment, the present invention providestetrahydropyrvinium (THP), derivatives thereof, benzoxazole compounds,and derivatives thereof.

In a sixth embodiment, the present invention provides a method of usingtetrahydropyrvinium (THP), derivatives thereof, benzoxazole compounds,and derivatives thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Compound screening strategy. Novel inhibitors of AR conformationchange were discovered by creating a HEK-293 (ATCC CRL-1573)-derivedcell line stably expressing a CFP N-terminal and YFP C-terminal taggedAR vector that has been previously used to measure conformation changeby FRET. C-AR-Y was stably expressed in either LAPC4 or HEK293 celllines; in parallel, HEK293 cells were transfected with native AR alongwith MMTV-luciferase. Cells were cultured in the presence or absence of10 nM dihydrotestosterone (DHT) and one of 1040 FDA-approved drugs. Thescreen was performed in duplicate for each condition, and the top 50compounds with activities in both trials were selected. Each of the topcompounds was evaluated in detail with a dose-response study, and the“validated” compounds were compared across all assays. The validatedhits accounted for approximately 40% of the hits initially identifiedfor each screen.

FIG. 2. Analysis of hits. 1040 FDA-approved drugs were tested in threedifferent assays, a transcription-based assay using HEK293 cellsexpressing MMTV-luciferase, conformation-based assays usingHEK293/C-AR-Y stable cells, and LAPC4/C-AR-Y stable cells. Fewercompounds affected AR conformational change vs. transcriptional output.LAPC4 cells were the least sensitive to test compounds. High selectivity(<50 hits) was observed for the transcription assay when the cutoff wasset between 5 and 6 standard deviations (SD) from the mean (asdetermined by multiple replicates of cells treated only with DHT).Similar selectivity was observed for the HEK293/C-AR-Y cells between 3and 4 SD, whereas in the LAPC4/C-AR-Y cells such effects were observedbetween 2 and 3 SD from the mean. The transcriptional assay used in thescreen had a Z-factor=0.6, vs. 0.6 for the LAPC4 FRET assay and 0.5 forthe HEK293 FRET assay (Zhang, J. H., T. D. Chung, and K. R. Oldenburg, ASimple Statistical Parameter for Use in Evaluation and Validation ofHigh Throughput Screening Assays. J Biomol Screen, 1999. 4(2): p.67-73).

FIG. 3. Characterization of hits in primary screen. (A) Hits that scoredin the primary assays were ranked according to efficacy based on theaverage of duplicate readings, and the top 50 from each assay wascompared to the other two. In each case, a minority of compounds wereshared between the assays, and most hits were unique to a particularsystem. (B) Validated hits were determined by detailed dose-response inthe original assay used, and only compounds that exhibited pharmacologiceffects were counted. These hits were then cross-compared to the otherscreening assays, using a dose-response. In this secondary analysis, themajority of hits from any one assay were also effective in another assaysystem.

FIG. 4. Pyrvinium pamoate (PP) exhibits identical responses vs.pyrvinium chloride (PCl). Anion exchange was used to replace the pamoatesalt with a chloride ion. The resulting compound, PCl, had an identicaldose-response vs. the parent compound in blocking PSA reporter activityin LAPC4 cells.

FIG. 5. PP and HH inhibit DHT-induced gene expression differently than acompetitive antagonist. LNCaP cells transiently transfected with aPSA-luciferase reporter were exposed to OH—F, PP and HH. DHT wastitrated. Whereas OH—F caused a modified DHT dose-response consistentwith a competitive antagonist, PP and HH exhibited a pattern consistentwith a non-competitive antagonist.

FIG. 6. PP and HH inhibit androgen-induced cell proliferation. PP, HH,and BiC were compared for their ability to inhibit androgen dependentand independent growth in several cultured cell lines: LNCaP, LAPC4,LN-AR (a line that exhibits androgen-independent growth), and HEK293cells. PP and HH each exhibited growth-inhibitory properties in LNCaPcells, whereas HH was not effective in LAPC4 cells. Neither compoundexhibited non-specific growth inhibition of HEK293 cells. Importantly,PP blocked growth of “hormone refractory” LN-AR cells. Asterisk(*)=p<0.005.

FIG. 7. PP synergistically reduces prostate size in mice. Cohorts ofnine male mice were treated with PO gavage of BiC (100 mg/kg), IPinjection of PP (1 mg/kg), or the combination for four weeks. As apositive control, nine mice were treated with castration for four weeks.Prostate glands were harvested and wet weights determined. PP alone didnot significantly reduce prostate size. BiC treatment significantlyreduced prostate weight by 35%, and the combination of PP:BiC reducedthe weight by 63% (p<0.0005, t-test), implying a synergistic effect ofPP. cntrl: untreated mice; BiC: bicalutamide; PP: pyrvinium pamoate;cast: castrated. Error bars represent the standard error of the mean(S.E.M.).

FIG. 8. PP suppresses androgen-dependent gene expression in theprostate, and augments BiC activity. Total RNA was extracted fromprostate glands of cohorts of 9 mice used to test PP in vivo. qRT-PCRwas performed to assess gene expression levels of five androgen-inducedgenes. Gene expression levels are expressed relative to RPL19, anandrogen-unresponsive gene. PP significantly suppressed gene expressionin all cases, and augmented the effects of BiC, with one exception(TMPRSS2), which may have been maximally suppressed by each treatmentalone. cntrl: untreated; BiC: bicalutamide; PP: pyrvinium pamoate; cast:castrated.

FIG. 9. Inhibitors of the androgen receptor.

FIG. 10. Inhibitors of the androgen receptor.

FIG. 11. Change in prostate wet weight after treatment with BiC, PP,THP+BiC, or castration.

FIG. 12. Histology of mice dorsal prostate after treatment with THP,BiC, THP+BiC, or castration.

FIG. 13. Quantitative PCR of androgen-regulated genes TMPRSS2 probasin,and fkbp51.

FIG. 14. THP efficacy against AR transcription in LAPC4 cells.

FIG. 15. Synergistic effects of THP+BiC against AR transcription inLAPC4 cells.

FIG. 16. THP and SHP efficacy against PSA-luciferase AR-responsivepromoter in LAPC4 cells.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention provides a method of inhibiting an androgenreceptor by administering to a patient in need of such treatment, atherapeutically effective amount of a compound of Formula I:

or a compound of Formula II:

The compounds of the present invention are believed to inhibit foldingof the androgen receptor, thus inhibiting receptor activation. Thecompounds of the present invention can be used to treat any diseaseinvolving folding of the androgen receptor. Patients in need of suchtreatment often suffer from prostate cancer, including primary andhormone refractory prostate cancer, ovarian cancer, hepatocellularcarcinoma, acne vulgaris, endometriosis, acanthosis nigricans,hypertrichosis, breast cancer, precocious puberty, polycystic ovarysyndrome, benign prostatic hyperplasia, alopecia (such asandrogen-dependent alopecia), hirsutism and hypersexuality/paraphilia.

The compounds of the present invention can be used to inhibit othernuclear receptors and treat associated disease states. Receptoractivation of PPARγ can be inhibited using the compounds of the presentinvention, thereby treating disease states such as insulin resistance,diabetes and lipodystrophy, including cholesterol disorders. Thecompounds of the present invention are useful in treating disease statesassociated with estrogen receptor α and β, such as breast, colon,ovarian and endometrial cancers, as well as in metabolic regulation.Other disease states that can be treated with the compounds of thepresent invention include those associated with the thyroid hormonereceptor, such as thyroid and cardiac disorders. Such compounds can alsobe used to augment the inhibition of the glucocorticoid receptor, whichis used for immune suppression in a multitude of diseases. The compoundsof the present invention can also inhibit the progesterone receptor,resulting in termination of a pregnancy.

II. Definitions

As used herein, “administering” refers to oral administration,administration as a suppository, topical contact, parenteral,intravenous, intraperitoneal, intramuscular, intralesional, intranasalor subcutaneous administration, intrathecal administration, or theimplantation of a slow-release device e.g., a mini-osmotic pump, to thesubject.

As used herein, the term “alkyl” refers to a straight or branched,saturated, aliphatic radical having the number of carbon atomsindicated. For example, C₁-C₆ alkyl includes, but is not limited to,methyl, ethyl, propyl, butyl, pentyl, hexyl, iso-propyl, iso-butyl,sec-butyl, tert-butyl, etc.

The term “lower” referred to above and hereinafter in connection withorganic radicals or compounds respectively defines a compound or radicalwhich can be branched or unbranched with up to and including 7,preferably up to and including 4 and (as unbranched) one or two carbonatoms.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be a variety of groups selected from: R′, —OR′,═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′,—CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NR—C(NR′R″)═NR′″, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′,—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —CN and —NO₂ in a number ranging fromzero to (2 m′+1), where m′ is the total number of carbon atoms in suchradical. R′, R″ and R′″ each independently refer to hydrogen,unsubstituted (C₁-C₈) alkyl and heteroalkyl, unsubstituted aryl, arylsubstituted with 1-3 halogens, unsubstituted alkyl, alkoxy or thioalkoxygroups, or aryl-(C₁-C₄)alkyl groups. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that substitutedalkyl is meant to include groups such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike). Preferably, substituted alkyl and heteroalkyl groups have from 1to 4 substituents; more preferably, 1, 2 or 3 substituents. Exceptionsare those perhalo alkyl groups (e.g., pentafluoroethyl and the like)which are also preferred and contemplated by the present invention.

Alternatively, R′, R″, R′″ and R″″ each preferably independently referto hydrogen, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl, e.g., aryl substituted with 1-3 halogens,substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, orarylalkyl groups. When a compound of the invention includes more thanone R group, for example, each of the R groups is independently selectedas are each R′, R″, R′″ and R″″ groups when more than one of thesegroups is present. When R′ and R″ are attached to the same nitrogenatom, they can be combined with the nitrogen atom to form a 5-, 6-, or7-membered ring. For example, —NR′R″ is meant to include, but not belimited to, 1-pyrrolidinyl and 4-morpholinyl.

As used herein, the term “alkylene” refers to an alkyl group linking atleast two other groups, i.e. a divalent hydrocarbon radical of 1 to 6carbon atoms. As for alkyl, the alkylene group can be straight orbranched. For instance, a straight chain alkylene can be the bivalentradical of —(CH₂)_(n)—, where n is 1, 2, 3, 4, 5 or 6. Alkylene groupsinclude, but are not limited to, methylene, ethylene, propylene,butylene, pentylene and hexylene. Similarly, “alkenylene,” “alkynylene”and “cycloalkylene” are divalent radicals of alkenyl, alkynyl andcycloalkyl (see within).

As used herein, the term “alkenyl” refers to either a straight chain orbranched hydrocarbon of 2 to 6 carbon atoms, having at least onecarbon-carbon double bond. Examples of alkenyl groups include, but arenot limited to, vinyl, propenyl, isopropenyl, butenyl, isobutenyl,butadienyl, pentenyl or hexadienyl.

As used herein, the term “alkynyl” refers to either a straight chain orbranched hydrocarbon of 2 to 6 carbon atoms, having at least onecarbon-carbon triple bond. Examples of alkynyl groups include, but arenot limited to, acetylenyl, propynyl or butynyl.

As used herein, the term “alkoxy” refers to alkyl with the inclusion ofan oxygen atom, for example, methoxy, ethoxy, etc.“Halo-substituted-alkoxy” is as defined for alkoxy wherein some or allof the hydrogen atoms are replaced with halogen atoms. For example,halo-substituted-alkoxy includes trifluoromethoxy, etc.

As used herein, the term “cycloalkyl” refers to a saturated or partiallyunsaturated, monocyclic, fused bicyclic, or bridged polycyclic ringassembly containing from 3 to 12 ring atoms (i.e., ring members; thatis, atoms directly connected to form the framework of the ring, such asthe six carbons in a cyclohexyl group), or the number of atoms indicatedFor example, C₃₋₈ cycloalkyl includes cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, and up to cyclooctyl.

As used herein, the term “heterocycloalkyl” refers to a ring systemhaving from 3 ring members to about 20 ring members and from 1 to about5 heteroatoms such as N, O and S. Additional heteroatoms can also beuseful, including, but not limited to, B, Al, Si and P. The heteroatomscan also be oxidized, such as, but not limited to, —S(O)— and —S(O)₂—.For example, heterocycloalkyl includes, but is not limited to,tetrahydrofuranyl, tetrahydrothiophenyl, morpholino, pyrrolidinyl,pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, piperidinyl, indolinyl, quinuclidinyl and1,4-dioxa-8-aza-spiro[4.5]dec-8-yl.

As used herein, the term “aryl” refers to a monocyclic or fusedbicyclic, tricyclic or greater, aromatic ring assembly containing 6 to16 ring carbon atoms. For example, aryl may be phenyl, benzyl ornaphthyl, preferably phenyl. “Arylene” means a divalent radical derivedfrom an aryl group. Aryl groups can be mono-, di- or tri-substituted byone, two or three radicals selected from alkyl, alkoxy, aryl, hydroxy,halogen, cyano, amino, amino-alkyl, trifluoromethyl, alkylenedioxy, andoxy-C₂-C₃-alkylene; or 1- or 2-naphthyl; or 1- or 2-phenanthrenyl.“Alkylenedioxy” is a divalent substitute attached to two adjacent carbonatoms of phenyl, e.g., methylenedioxy or ethylenedioxy.“Oxy-C₂-C₃-alkylene” is also a divalent substituent attached to twoadjacent carbon atoms of phenyl, e.g., oxyethylene or oxypropylene. Anexample for oxy-C₂-C₃-alkylene-phenyl is 2,3-dihydrobenzofuran-5-yl.

Preferred aryl groups include naphthyl, phenyl, or phenyl mono- ordisubstituted by alkoxy, phenyl, halogen, alkyl, or trifluoromethyl;more preferably, phenyl or phenyl mono- or disubstituted by alkoxy,halogen or trifluoromethyl; and, phenyl.

Examples of substituted phenyl groups as R are, e.g., 4-chlorophen-1-yl,3,4-dichlorophen-1-yl, 4-methoxyphen-1-yl, 4-methylphen-1-yl,4-aminomethylphen-1-yl, 4-methoxyethylaminomethylphen-1-yl,4-hydroxyethylaminomethylphen-1-yl,4-hydroxyethyl-(methyl)-aminomethylphen-1-yl, 3-aminomethylphen-1-yl,4-N-acetylaminomethylphen-1-yl, 4-aminophen-1-yl, 3-aminophen-1-yl,2-aminophen-1-yl, 4-phenyl-phen-1-yl, 4-(imidazol-1-yl)-phenyl,4-(imidazol-1-ylmethyl)-phen-1-yl, 4-(morpholin-1-yl)-phen-1-yl,4-(morpholin-1-ylmethyl)-phen-1-yl,4-(2-methoxyethylaminomethyl)-phen-1-yl and4-(pyrrolidin-1-ylmethyl)-phen-1-yl, 4-(thiophenyl)-phen-1-yl,4-(3-thiophenyl)-phen-1-yl, 4-(4-methylpiperazin-1-yl)-phen-1-yl, and4-(piperidinyl)-phenyl and 4-(pyridinyl)-phenyl.

Similarly, other substituents for the aryl and heteroaryl groups arevaried and are selected from: -halogen, —OR′, —OC(O)R′, —NR′R″, —SR′,—R′, —CN, —NO₂, —CO₂R′, —CONR′R″, —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′,—NR″C(O)₂R′, —NR′—C(O)NR″R′″, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH,—NH—C(NH₂)═NR′, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —N₃, —CH(Ph)₂,perfluoro(C₁-C₄)alkoxy, and perfluoro(C₁-C₄)alkyl, in a number rangingfrom zero to the total number of open valences on the aromatic ringsystem; and where R′, R″ and R′″ are independently selected fromhydrogen, (C₁-C₈)alkyl and heteroalkyl, unsubstituted aryl andheteroaryl, (unsubstituted aryl)-(C₁-C₄)alkyl, and (unsubstitutedaryl)oxy-(C₁-C₄)alkyl.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CH₂)_(q)—U—, wherein T and U are independently —NH—, —O—, —CH₂—or a single bond, and q is an integer of from 0 to 2. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula-A-(CH₂)_(r)—B—, wherein A and B are independently —CH₂—, —O—, —NH—,—S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is an integerof from 1 to 3. One of the single bonds of the new ring so formed mayoptionally be replaced with a double bond. Alternatively, two of thesubstituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CH₂)_(s)—X—(CH₂)_(t)—, where s and t are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituent R′ in —NR′— and —S(O)₂NR′— is selected from hydrogen orunsubstituted (C₁-C₆)alkyl.

As used herein, the term “heteroaryl” refers to a monocyclic or fusedbicyclic or tricyclic aromatic ring assembly containing 5 to 16 ringatoms (i.e., ring members; that is, atoms directly connected to form theframework of the ring, such as the five carbons and one nitrogen in a2-pyridyl group), where from 1 to 4 of the ring atoms are a heteroatomeach N, O or S. For example, heteroaryl includes pyridyl, indolyl,indazolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl,benzofuranyl, furanyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl,isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, orany other radicals substituted, especially mono- or di-substituted, byalkyl, nitro, or halogen. Pyridyl represents 2-, 3- or 4-pyridyl,advantageously 2- or 3-pyridyl. Thienyl represents 2- or 3-thienyl.Quinolinyl represents preferably 2-, 3- or 4-quinolinyl. Isoquinolinylrepresents preferably 1-, 3- or 4-isoquinolinyl. Benzopyranyl,benzothiopyranyl represents preferably 3-benzopyranyl or3-benzothiopyranyl, respectively. Thiazolyl represents preferably 2- or4-thiazolyl, and most preferred, 4-thiazolyl. Triazolyl is preferably1-, 2- or 5-(1,2,4-triazolyl). Tetrazolyl is preferably 5-tetrazolyl.

Preferably, heteroaryl is pyridyl, indolyl, quinolinyl, pyrrolyl,thiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl,thienyl, furanyl, benzothiazolyl, benzofuranyl, isoquinolinyl,benzothienyl, oxazolyl, indazolyl, or any of the radicals substituted,especially mono- or di-substituted by alkyl, nitro, or halogen.

As used herein, the term “androgen receptor” refers to an intracellularsteroid receptor of the nuclear receptor super-family that specificallybinds androgens such as testosterone and dihydrotestosterone.

As used herein, the term “anti-androgen” refers to a group of hormonereceptor antagonist compounds that are capable of preventing orinhibiting the biologic effects of androgens, male sex hormones, onnormally responsive tissues in the body. Antiandrogens usually work byblocking the appropriate receptors, competing for binding sites onintracellular receptors, and obstructing androgen signaling pathways. Aswell as the compounds of Formula I and II, anti-androgens include, butare not limited to, coumarins, bicalutamide, flutamide,hydroxyflutamide, nilutamide, spionolactone, cyproterone acetate,ketoconazole, finasteride, dutasteride, harman, norharman, harmine,harmaline, tetrahydroharmine, harmol, harmalol, ethyl harmol, n-butylharmol and other beta-carboline derivatives.

Antiandrogens are often indicated to treat severe male sexual disorders,such as hypersexuality (excessive sexual desire) and sexual deviation,specifically paraphilias, as well as use as an antineoplastic agent andpalliative, adjuvant or neoadjuvant hormonal therapy in prostate cancer.Antiandrogens can also be used for treatment of benign prostatichyperplasia (prostate enlargement), acne vulgaris, androgenetic alopecia(male pattern baldness), and hirsutism (excessive hair growth).Anti-androgens are also occasionally used as a male contraceptive agent,to purposefully prevent or counteract masculinisation in the case oftransgender women undergoing gender reassignment therapy, and to preventthe symptoms associated with reduced testosterone, such as hot flashes,following castration. Other conditions treatable with an anti-androgenare prostate cancer, including primary and hormone refractory prostatecancer, ovarian cancer, hepatocellular carcinoma, acne vulgaris,endometriosis, acanthosis nigricans, hypertrichosis, breast cancer,precocious puberty, polycystic ovary syndrome, benign prostatichyperplasia, alopecia (such as androgen-dependent alopecia), hirsutismand hypersexuality/paraphilia.

As used herein, the term “a combination of active agents” refers to acomposition of at least two or more active agents.

As used herein, the term “counterion” refers to the ion that accompaniesan ionic species in order to maintain electronic neutrality. Counterionscan be atomic, such as fluoride, chloride, bromide, iodide, or metalliccounterions. Counterions can also be molecular, such as acetate,succinate, maleate and embonate (pamoate). Counterions can be positivelyor negatively charged. Counterions of the present invention arenegatively charged. In addition, counterions can have a charge greaterthan 1, such as 2 or more. One of skill in the art will appreciate thatother counterions are useful in the present invention.

As used herein, the term “hormonal therapy” refers to the use ofhormones in medical treatment, as well as the inhibition of hormoneproduction, such as the use of direct competitors to hormones, such asantiandrogens.

As used herein, the term “hydrate” refers to a compound that iscomplexed to at least one water molecule. The compounds of the presentinvention can be complexed with from 1 to 10 water molecules.

As used herein, the term “inhibiting” refers to a compound thatpartially or fully prohibits or a method of partially or fullyprohibiting a specific action or function.

As used herein, the term “LnRH agonist” refers to a compound orbiological molecule that binds to the luteinizing releasing-hormonereceptor.

As used herein, the term “patient in need” refers to a patient sufferingfrom prostate cancer, polycystic ovary syndrome, benign prostatichyperplasia, alopecia and hirsutism. Other conditions that a patient inneed suffers from include, but are not limited to, ovarian cancer,hepatocellular carcinoma, acne vulgaris, endometriosis, acanthosisnigricans, hypertrichosis, breast cancer, precocious puberty andhypersexuality/paraphilia. Patients suffering from other conditionstreatable with anti-androgens are also treatable with the methods of thepresent invention. Patients treatable using the methods of the presentinvention are animals such as mammals, including, but not limited to,primates (e.g., humans), cows, sheep, goats, horses, dogs, cats,rabbits, rats, mice and the like. In certain embodiments, the patient isa human.

As used herein, the term “prodrug” refers to covalently bonded carrierswhich are capable of releasing the active agent of the methods of thepresent invention, when the prodrug is administered to a mammaliansubject. Release of the active ingredient occurs in vivo. Prodrugs canbe prepared by techniques known to one skilled in the art. Thesetechniques generally modify appropriate functional groups in a givencompound. These modified functional groups however regenerate originalfunctional groups by routine manipulation or in vivo. Prodrugs of theactive agents of the present invention include active agents wherein ahydroxy, amidino, guanidino, amino, carboxylic or a similar group ismodified.

As used herein, the term “salt” refers to acid or base salts of thecompounds used in the methods of the present invention. Illustrativeexamples of pharmaceutically acceptable salts are mineral acid(hydrochloric acid, hydrobromic acid, phosphoric acid, and the like)salts, organic acid (acetic acid, propionic acid, glutamic acid, citricacid and the like) salts, quaternary ammonium (methyl iodide, ethyliodide, and the like) salts. It is understood that the pharmaceuticallyacceptable salts are non-toxic. Additional information on suitablepharmaceutically acceptable salts can be found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, which is incorporated herein by reference.

Pharmaceutically acceptable salts of the basic compounds of the presentinvention are salts formed with acids, such as of mineral acids, organiccarboxylic and organic sulfonic acids, e.g., hydrochloric acid,methanesulfonic acid, maleic acid, are also possible provided a basicgroup, such as pyridyl, constitutes part of the structure.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

As used herein, the terms “therapeutically effective amount or dose” or“therapeutically sufficient amount or dose” or “effective or sufficientamount or dose” refer to a dose that produces therapeutic effects forwhich it is administered. The exact dose will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms(vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999);and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003,Gennaro, Ed., Lippincott, Williams & Wilkins). In sensitized cells, thetherapeutically effective dose can often be lower than the conventionaltherapeutically effective dose for non-sensitized cells.

As used herein, the terms “treat”, “treating” and “treatment” refers toany indicia of success in the treatment or amelioration of an injury,pathology, condition, or symptom (e.g., pain), including any objectiveor subjective parameter such as abatement; remission; diminishing ofsymptoms or making the symptom, injury, pathology or condition moretolerable to the patient; decreasing the frequency or duration of thesymptom or condition; or, in some situations, preventing the onset ofthe symptom or condition. The treatment or amelioration of symptoms canbe based on any objective or subjective parameter; including, e.g., theresult of a physical examination.

III. Method of Inhibiting an Androgen Receptor

The present invention provides a method of inhibiting an androgenreceptor by administering to a patient in need of such treatment, atherapeutically effective amount of a compound of Formula I, Formula II,or compounds shown in FIG. 9 and FIG. 10. Compounds useful in themethods of the present invention include compounds of Formula I:

and compounds of Formula II:

wherein each R¹ is independently hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl,C₂₋₆ alkynyl, C₁₋₆ alkoxy, —OR⁴, —SR⁴, —NR⁴R⁵, cycloalkyl,heterocycloalkyl, aryl or heteroaryl. R² is hydrogen, C₁₋₆ alkyl, C₁₋₆alkyl-OH, C₂₋₆ alkenyl or C₂₋₆ alkynyl. R^(2′) is an electron pair,hydrogen, C₁₋₆ alkyl, C₁₋₆ alkyl-OH, C₂₋₆ alkenyl or C₂₋₆ alkynyl. R³ iscycloalkyl, heterocycloalkyl, aryl or heteroaryl, optionally substitutedwith from 1 to 3 R⁶ groups. R⁴ and R⁵ are each independently hydrogen,C₁₋₆ alkyl, C₂₋₆ alkenyl or C₂₋₆ alkynyl. Alternatively R⁴ and R⁵ arecombined with the nitrogen to which they are attached to form aheterocyclic ring having from 5 to 7 ring members and from 1 to 3heteroatoms each independently N, O or S. Each R⁶ is independently H,C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl or C₁₋₆ alkoxy. L is a linker ofC₁₋₆ alkylene, C₂₋₆ alkenylene, C₂₋₆ alkynylene or C₃₋₆ cycloalkylene. Xis —N(R⁷)—, an aryl ring having 6-10 ring members and a heteroaryl ringhaving from 5 to 6 ring members and from 1 to 3 heteroatoms eachindependently N, O or S, wherein the aryl and the heteroaryl ring areeach optionally substituted with from 1 to 3 R⁸ groups. R⁷ is H, C₁₋₆alkyl, C₁₋₆ alkenyl or C₂₋₆ alkynyl. Each R⁸ is independently H, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl or C₁₋₆ alkoxy. Y is O or S. Thecompounds of Formulas I and II include the salts, hydrates and prodrugsthereof. By administering the compound of Formula I, the method inhibitsthe androgen receptor.

Some of the compounds of Formulas I and II are already known. Othercompounds of Formulas I and II are novel compounds. The compounds ofFormulas I and II can be combined with other anti-androgen compoundssuch as coumarins, bicalutamide, flutamide, hydroxyflutamide,nilutamide, spionolactone, cyproterone acetate, ketoconazole,finasteride, dutasteride, harman, harmine, harmaline, tetrahydroharmine,harmol, harmalol, harmine acid, harmine acid methyl ester, harmilinicacid, harmanamide, acetylnorharmine, ethyl harmol, n-butyl harmol andother beta-carboline derivatives.

The compounds of the present invention are believed to inhibit foldingof the androgen receptor, thus inhibiting receptor activation. Patientsin need of such treatment often suffer from prostate cancer, includingprimary and hormone refractory prostate cancer, ovarian cancer,hepatocellular carcinoma, acne vulgaris, endometriosis, acanthosisnigricans, hypertrichosis, breast cancer, precocious puberty, polycysticovary syndrome, benign prostatic hyperplasia, alopecia (such asandrogen-dependent alopecia), hirsutism and hypersexuality/paraphilia.Other disease states can be treated using the methods of the presentinvention.

The compounds of the present invention are believed to inhibit foldingof the androgen receptor, thus inhibiting receptor activation. Patientsin need of such treatment often suffer from prostate cancer, includingprimary and hormone refractory prostate cancer, ovarian cancer,hepatocellular carcinoma, acne vulgaris, endometriosis, acanthosisnigricans, hypertrichosis, breast cancer, precocious puberty, polycysticovary syndrome, benign prostatic hyperplasia, alopecia (such asandrogen-dependent alopecia), hirsutism and hypersexuality/paraphilia.Other disease states can be treated using the methods of the presentinvention.

In another embodiment, the method of the present invention treatsalopecia by topical administration of a compound or composition of thepresent invention.

In other embodiments, the compounds of Formula I and Formula II areadministered with a course of hormonal therapy, where the compound forhormonal therapy is an anti-androgen or a LnRH agonist. In someembodiments, the compounds are administered separately. In otherembodiments, the compounds are admixed. In some other embodiments, thecompounds are administered at the same time. In still other embodiments,the compounds are administered at different times.

In some other embodiments, the compounds of Formula I and Formula II areadministered in combination with a therapeutically effective amount ofdocetaxel (taxol), paclitaxel (taxotere), bicalutamide, flutamide,hydroxyflutamide, nilutamide, spionolactone, cyproterone acetate,ketoconazole, finasteride or dutasteride. In other embodiments, thecompounds of Formula I and Formula II are administered in combinationwith a therapeutically effective amount of a coumarin.

IV. Compounds for Inhibiting an Androgen Receptor

Compounds useful in the present invention are those that inhibit anandrogen receptor. Useful compounds can be identified using the assaymethods described in the Examples below. These compounds arecommercially available or can be synthesized using methods known tothose skilled in the art.

In some embodiments, compounds useful in the methods of the presentinvention include compounds of Formula I:

wherein each R¹ is independently hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl,C₂₋₆ alkynyl, C₁₋₆ alkoxy, —OR⁴, —SR⁴, —NR⁴R⁵, cycloalkyl,heterocycloalkyl, aryl or heteroaryl. R² is hydrogen, C₁₋₆ alkyl, C₁₋₆alkyl-OH, C₂₋₆ alkenyl or C₂₋₆ alkynyl. R³ is cycloalkyl,heterocycloalkyl, aryl or heteroaryl, optionally substituted with from 1to 3 R⁶ groups. R⁴ and R⁵ are each independently hydrogen, C₁₋₆ alkyl,C₂₋₆ alkenyl or C₂₋₆ alkynyl. Alternatively R⁴ and R⁵ are combined withthe nitrogen to which they are attached to form a heterocyclic ringhaving from 5 to 7 ring members and from 1 to 3 heteroatoms eachindependently N, O or S. Each R⁶ is independently H, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl or C₁₋₆ alkoxy. L is a linker of C₁₋₆ alkylene,C₂₋₆ alkenylene, C₂₋₆ alkynylene or C₃₋₆ cycloalkylene. X is —N(R⁷)—, anaryl ring having 6-10 ring members and a heteroaryl ring having from 5to 6 ring members and from 1 to 3 heteroatoms each independently N, O orS, wherein the aryl and the heteroaryl ring are each optionallysubstituted with from 1 to 3 R⁸ groups. R⁷ is H, C₁₋₆ alkyl, C₂₋₆alkenyl or C₂₋₆ alkynyl. Each R⁸ is independently H, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl or C₁₋₆ alkoxy. The compounds of Formula I includethe salts, hydrates and prodrugs thereof.

In some embodiments, L is ethylene, ethenylene or cyclopropylene.

In some other embodiments, compounds of the present invention includecompounds of Formula Ia:

In other embodiments, X is heteroaryl. In still other embodiments, X ispyrrole. In yet other embodiments, X is aryl. In still yet otherembodiments, the compound can be:

In other embodiments, compounds of the present invention includecompounds of the Formula:

wherein R¹ is —NR⁴R⁵, such that R⁴ and R⁵ are combined with the nitrogento which they are attached to form a heterocyclic ring having from 5 to7 ring members and from 1 to 3 heteroatoms each independently N, O or S.R³ is aryl. X is heteroaryl.

In some embodiments, the compounds of the present invention includecompounds of Formula Ib:

In other embodiments, the compound of Formula Ib is:

In some other embodiments, the compounds of the present inventioninclude compounds of Formula Ic:

In another embodiment, the compounds of the present invention includecompounds of Formula Id:

In other embodiments, salt forms of the compounds of Formula I include acounterion of pamoate, chloride, bromide, succinate, maleate or acetate.

In other embodiments, compounds useful in the methods of the presentinvention include compounds of Formula II:

wherein R¹, R³, R⁴, R⁵, R⁶, R⁷, R⁸, X and L are as defined above; R^(2′)is an electron pair, hydrogen, C₁₋₆ alkyl, C₁₋₆ alkyl-OH, C₂₋₆ alkenylor C₂₋₆ alkynyl; and Y is O or S. The compounds of Formula II includethe salts, hydrates and prodrugs thereof.

In some embodiments, the compounds of Formula II include the following:

wherein X is aryl or heteroaryl. In other embodiments, the compoundshave the following formula:

In some other embodiments, le and R³ are both aryl, and le and R³ areboth C₁₋₆ alkyl. In still other embodiments, the compound is:

V. Formulations for Inhibiting an Androgen Receptor

The compounds of the present invention can be formulated in a variety ofdifferent manners known to one of skill in the art. Pharmaceuticallyacceptable carriers are determined in part by the particular compositionbeing administered, as well as by the particular method used toadminister the composition. Accordingly, there are a wide variety ofsuitable formulations of pharmaceutical compositions of the presentinvention (see, e.g., Remington's Pharmaceutical Sciences, 20^(th) ed.,2003, supra). For example, the compounds of the present invention can beprepared and administered in a wide variety of oral, injectable andtopical dosage forms. The compounds of the present invention can also beprepared and administered in parenteral dosage forms. Thus, thecompounds of the present invention can be administered by injection,that is, intravenously, intramuscularly, intracutaneously,subcutaneously, intraduodenally, or intraperitoneally. Also, thecompounds described herein can be administered by inhalation, forexample, intranasally. Additionally, the compounds of the presentinvention can be administered transdermally or topically, e.g., in aliquid or gel form or as a patch.

Accordingly, the present invention also provides pharmaceuticalcompositions comprising a pharmaceutically acceptable carrier orexcipient and one or more compounds of the invention. In someembodiments, the compound can be any of the following:

In other embodiments, the compound can be:

Formulations suitable for administration can consist of (a) liquidsolutions, such as an effective amount of a compound of the presentinvention suspended in diluents, such as water, saline or PEG 400; (b)capsules, sachets, depots or tablets, each containing a predeterminedamount of the active ingredient, as liquids, solids, granules orgelatin; (c) suspensions in an appropriate liquid; (d) suitableemulsions; and (e) patches. The pharmaceutical forms can include one ormore of lactose, sucrose, mannitol, sorbitol, calcium phosphates, cornstarch, potato starch, microcrystalline cellulose, gelatin, colloidalsilicon dioxide, talc, magnesium stearate, stearic acid, and otherexcipients, colorants, fillers, binders, diluents, buffering agents,moistening agents, preservatives, flavoring agents, dyes, disintegratingagents, and pharmaceutically compatible carriers. Lozenge forms cancomprise the active ingredient in a flavor, e.g., sucrose, as well aspastilles comprising the active ingredient in an inert base, such asgelatin and glycerin or sucrose and acacia emulsions, gels, and the likecontaining, in addition to the active ingredient, carriers known in theart.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form. The composition can, if desired, also contain othercompatible therapeutic agents. Preferred pharmaceutical preparations candeliver the compounds of the invention in a sustained releaseformulation.

The pharmaceutical preparations are typically delivered to a mammal,including humans and non-human mammals. Non-human mammals treated usingthe present methods include domesticated animals (i.e., canine, feline,murine, rodentia, and lagomorpha) and agricultural animals (bovine,equine, ovine, porcine).

For cancer therapy, formulations of the present invention can include acompound of Formulas I or II in combination with a therapeuticallyeffective amount of an anti-androgen or an LnRH agonist. Anti-androgens,as described above, include, but are not limited to, coumarins,bicalutamide, flutamide, hydroxyflutamide, nilutamide, spionolactone,cyproterone acetate, ketoconazole, finasteride, dutasteride, harman,norharman, harmine, harmaline, tetrahydroharmine, harmol, harmalol,ethyl harmol, n-butyl harmol and other beta-carboline derivatives.

In practicing the methods of the present invention, the pharmaceuticalcompositions can be used alone, or in combination with other therapeuticor diagnostic agents. The additional anticancer drugs used in thecombination protocols of the present invention can be formulatedseparately, or one or more of the anticancer drugs used in thecombination protocols can be formulated together, such as in anadmixture.

Formulations of the present invention can also include combinations ofcompounds of Formulas I and II. Additional therapeutic agents ordiagnostic agents, such as those provided above, can also be formulatedin combination with the combination of compounds of Formulas I and II.

VI. Administration to Inhibit an Androgen Receptor

The compounds of the present invention can be administered as frequentlyas necessary, including hourly, daily, weekly or monthly. The compoundsutilized in the pharmaceutical method of the invention are administeredat the initial dosage of about 0.0001 mg/kg to about 1000 mg/kg daily. Adaily dose range of about 0.01 mg/kg to about 500 mg/kg, or about 0.1mg/kg to about 200 mg/kg, or about 1 mg/kg to about 100 mg/kg, or about10 mg/kg to about 50 mg/kg, can be used. The dosages, however, may bevaried depending upon the requirements of the patient, the severity ofthe condition being treated, and the compound being employed. Forexample, dosages can be empirically determined considering the type andstage of disease diagnosed in a particular patient. The doseadministered to a patient, in the context of the present inventionshould be sufficient to effect a beneficial therapeutic response in thepatient over time. The size of the dose also will be determined by theexistence, nature, and extent of any adverse side-effects that accompanythe administration of a particular compound in a particular patient.Determination of the proper dosage for a particular situation is withinthe skill of the practitioner. Generally, treatment is initiated withsmaller dosages which are less than the optimum dose of the compound.Thereafter, the dosage is increased by small increments until theoptimum effect under circumstances is reached. For convenience, thetotal daily dosage may be divided and administered in portions duringthe day, if desired. Doses can be given daily, or on alternate days, asdetermined by the treating physician. Doses can also be given on aregular or continuous basis over longer periods of time (weeks, monthsor years), such as through the use of a subdermal capsule, sachet ordepot, or via a patch.

The pharmaceutical compositions can be administered to the patient in avariety of ways, including topically, parenterally, intravenously,intradermally, intramuscularly, colonically, rectally orintraperitoneally. Preferably, the pharmaceutical compositions areadministered parenterally, topically, intravenously, intramuscularly ororally.

In practicing the methods of the present invention, the pharmaceuticalcompositions can be used alone, or in combination with other therapeuticor diagnostic agents. The additional anticancer drugs used in thecombination protocols of the present invention can be administeredseparately or one or more of the anticancer drugs used in thecombination protocols can be administered together, such as in anadmixture. Where one or more anticancer drug is administered separately,the timing and schedule of administration of each drug can vary. Theother therapeutic or diagnostic agents can be administered at the sametime as the compounds of Formulas I and II, separately or at differenttimes.

The compounds of Formulas I and II can also be administered in anysuitable combination. Additional therapeutic agents or diagnostic agentscan be administered in combination with the combination of Formulas Iand II.

The compounds of the present invention can be administered with a courseof hormonal therapy. The compound for hormonal therapy includes, but isnot limited to, an anti-androgen and a LnRH agonist.

In clinical studies, number of lesions, tumor size, and tumor growthrate can be monitored by radiography, tomography, and, where possible,direct measurement of tumor mass. Anti-tumor effects can also bemeasured using molecular biology and biochemistry techniques, such asELISA, PCR, western blotting, or immunocytochemistry.

The pharmaceutically effective amount of a composition required as adose will depend on the route of administration, the type of cancerbeing treated, and the physical characteristics of the patient. The dosecan be tailored to achieve a desired effect, but will depend on suchfactors as body surface area, weight, diet, concurrent medication andother factors which those skilled in the medical arts will recognize.

The foregoing are general guidelines only that can be expanded oraltered based on, for example, disease type and grade, patient age,health status, and sex, the particular drugs used in combination, theroute and frequency of administration, and experimental and clinicalfindings using a multidrug combination.

VII. Examples Example 1 Compound Preparation

Compounds useful in the methods of the present invention can be preparedfollowing the procedures set forth below. Pyrvinium pamoate iscommercially available from MP Biochemicals (Solon, Ohio). Harmol HCl iscommercially available from Sigma (St. Louis, Mo.).

{2-[2-(2,5-Dimethyl-1-phenyl-1H-pyrrol-3-yl)-vinyl]-1-methyl-1,2,3,4-tetrahydro-quinolin-6-yl}-dimethyl-amine

6-Dimethylamino-2-[2-(2,5-dimethyl-1-phenyl-1H-pyrrol-3-yl)-vinyl]-1-methyl-quinolinium,pamoate salt (pyrvinium pamoate, 500 mg, 0.434 mmol) was dissolved in 10ml of EtOH with stirring at room temperature. Sodium borohydride (67.5mg, 1.74 mmol) was added in one portion. Stirring was continued for 3.5hours until the reaction was judged complete by LC-MS analysis. Thereaction mixture was treated with water (10 ml) and extracted threetimes with dichloromethane. The dichloromethane layers were consolidatedand dried (sodium sulfate), filtered, and concentrated. The residue (394mg) was purified by high-performance flash chromatography (Biotage 25+M,silica cartridge) using a gradient of 0-25% ethyl acetate in hexane over12 column volumes to afford the{2-[2-(2,5-Dimethyl-1-phenyl-1H-pyrrol-3-yl)-vinyl]-1-methyl-1,2,3,4-tetrahydro-quinolin-6-yl}-dimethyl-amineas yellow-orange oil. Yield: 150 mg (45%). ¹H NMR: (400 MHz, CDCl₃) 1.90(m, 1H), 1.94 (s, 3H), 1.97, (s, 3H), 2.09 (m, 1H), 2.72-2.80 (m, 2H),2.79 (s, 6H), 2.83 (s, 3H), 3.74 (m, 1H), 5.78 (dd, J=8 Hz, 16 Hz, 1H),6.08 (s, 1H), 6.38 (d, J=16 Hz, 1H), 6.56 (m, 2H), 6.65 (m, 1H), 7.13(m, 2H), 7.38 (m, 3H) ESI-MS (m/z): [M+H]⁺=386.

Dimethyl-1-phenyl-1H-pyrrol-3-yl)-ethyl]-1-methyl-1,2,3,4-tetrahydro-quinolin-6-yl}-dimethyl-amine

{2-[2-(2,5-Dimethyl-1-phenyl-1H-pyrrol-3-yl)-vinyl]-1-methyl-1,2,3,4-tetrahydro-quinolin-6-yl}-dimethyl-amine,(25 mg, 0.065 mmol) was dissolved in 4 ml methanol and combined with 10%Pd/C (13 mg). The mixture was stirred under an atmosphere of hydrogengas (balloon) for 2 hrs or until the reaction was complete as judged byLC-MS analysis. The reaction mixture was then filtered through celite,washing with additional methanol, and the filtrate concentrated toafford an oil. The crude residue (27 mg) was purified by preparativeHPLC to afford the title compound,{2-[2-(2,5-Dimethyl-1-phenyl-1H-pyrrol-3-yl)-ethyl]-1-methyl-1,2,3,4-tetrahydro-quinolin-6-yl}-dimethyl-amineas a trifluoroacetate (TFA) salt. Yield: 23 mg (70%). ¹H NMR: (400 MHz,CDCl₃) 1.59 (m, 1H), 1.85-2.03 (m, 3H), 1.91 (s, 3H), 1.97 (s, 3H),2.33-2.52 (m, 2H), 2.67-2.92 (m, 2H), 2.94 (s, 3H), 3.11 (s, 6H),3.36-3.42 (m, 1H), 5.78 (s, 1H), 6.53 (d, J=8 Hz, 1H), 1.83 (m, 1H),7.08-7.39 (m, 3H), 7.32-7.46 (m, 3H) ESI-MS (m/z): [M+H]⁺=388.

Example 2 Screening for Novel AR Antagonists

The assay below provides a method of identifying compounds that areandrogen-receptor antagonists.

A library assembled by the NINDS was screened. The library consisted of1040 FDA-approved drugs and natural products. A basic strategy to selectand compare compounds from each of the primary screens was established(FIG. 1).

The transcription assay was conducted by transfecting 10 cm plates ofHEK293 cells with plasmids encoding MMTV-luciferase, SV40-renillaluciferase, and native AR. These cells are useful because the limitedtransfection efficiency of prostate-derived cells increases thevariability of the assay. The FRET assays were conducted using twoindependent cell lines, each stably expressing a CFP-AR-YFP fusionprotein: HEK293/C-AR-Y and LAPC4/C-AR-Y. LAPC4 cells are a prostatederived line (Klein, K. A., et al., Progression of metastatic humanprostate cancer to androgen independence in immunodeficient SCID mice.Nat Med, 1997. 3(4): p. 402-8). FRET is fluorescence resonance energytransfer between cyan and yellow fluorescent protein derivatives (CFP,YFP) that are fused to the amino and carboxyl termini of the humanandrogen receptor. When ligand activates the receptor, a conformationalchange takes place that brings the CFP and YFP into close proximity,thereby allowing FRET to occur. This spectroscopic change is detectedusing a fluorescence plate reader.

For the transcription assay, transfected cells were pooled before beingplated into 96-well dishes with the test compounds at 10 μM. On eachplate was included “no DHT” and “no treatment” controls, as well as apositive control with 1 μM OH—F. For the FRET assays, cells weredirectly plated into the 96-well plates with the test compounds at 10μM. Cell and compound transfers were accomplished with a liquid handlingrobot. Cells were cultured for 24 hours in the presence of 10 nM DHT andthe test compounds. After 24 hours, cells were either lysed byfreeze-thaw (transcription assay), or fixed in 4% paraformaldehyde forreading on the FPR (FRET assays). Each compound was tested in duplicatein the initial screen.

The compounds were sorted on the basis of anti-androgen effects. Inorder to be selected for further analysis, a test compound had tofunction in both of the replicates, and in each case had to reduce thesignal by at least one standard deviation from the mean of samplestreated with DHT alone. This eliminated situations in which one testgave a strong response, and the replicate had none. Lastly, there musthave been no evidence of cell toxicity, for the transcription assaysspecifically tested for renilla luciferase (which is discriminated fromfirefly luciferase on the basis of substrate specificity), and compoundsthat reduced this signal below two standard deviations were eliminated.For the FRET assays, toxic compounds were easily detected because oftheir effect on raw CFP and YFP signal, and compounds that reduced thissignal below two standard deviations were eliminated.

Analysis of Hits from the Primary Screen.

FIG. 2 illustrates the results of the screen. The data clearly indicatethat each assay system was capable of sorting compounds according toefficacy. Hits were sorted using different stringencies to determine thedegree of variance from the mean that would be required to select alimited number of hits for each assay. The conformational assay, whenused in either HEK293 or LAPC4 cells, achieved high selectivity using alower stringency, whereas the transcription-based assay required higherdegrees of stringency to achieve the same selectivity. Accordingly,conformational assays are less sensitive to non-specific cellularperturbation, whereas many compounds were capable of perturbing thetranscriptional response.

The top 50 compounds identified in each primary assay were then testedin the other two assays. Among the top hits in each assay that were notincluded for further analysis were all known anti-androgens from thelibrary (hydroxy-flutamide, nilutamide, cyproterone, and cyproteroneacetate), and certain steroid hormones that exhibit cross-reactivitywith AR at micromolar concentrations. A Venn diagram illustrates thatthere was modest overlap between the primary assays: approximately20-30% of hits that scored in one assay also scored in another assay(FIG. 3).

To determine “validated” hits, the top 50 compounds were re-tested withtitration studies in the original assays in which they were detected,and selected only those that exhibited a classic dose-response pattern.When validated hits from each assay were compared for activity in theothers, a much higher proportion of compounds (approximately 55-82%)scored positive in at least one other assay than in the primary screen.Few compounds (3) scored positive in all three assays. This isconsistent with the idea that each screening approach has the potentialto identify compounds with efficacy across assays, but that it alsomight add a new dimension to drug discovery. Importantly, among thesmall number of validated hits, it was discovered that one compound inthe HEK293 FRET assay, and three compounds in the LAPC4 FRET assay thatinitially did not meet selection criteria in the HEK293 primarytranscription assay, but which showed inhibition of AR-mediatedtranscription upon subsequent careful analysis. Accordingly, the FRETassays can augment drug discovery vs. transcription assays alone.

Identification of Novel Anti-Androgens.

Several lead compounds survived the stringent screening protocol, andthree classes with previously unrecognized anti-androgen activity: thecoumarins, including warfarin, scopoletin, and esculin; harmolhydrochloride, a natural product (HH); and an FDA-approvedanti-helminthic, pyrvinium pamoate (PP).

To evaluate the efficacy of pyrvinium and harmol against endogenous ARactivity, each compound was tested in a dose-response assay in twoprostate cancer-derived cell lines, LAPC4 (Klein, K. A., et al.,Progression of metastatic human prostate cancer to androgen independencein immunodeficient SCID mice. Nat Med, 1997. 3(4): p. 402-8), and LNCaP(Horoszewicz, J. S., et al., The LNCaP cell line—a new model for studieson human prostatic carcinoma. Prog Clin Biol Res, 1980. 37: p. 115-32)that each express endogenous AR. LAPC4 cells express wild-type AR,whereas LNCaP cells, which are derived from hormone-refractory PCa,express AR with a mutation (T877A) that renders it responsive to avariety of ligands, including the antagonist hydroxy-flutamide (OH—F).Cells were transfected with two reporter plasmids, a PSA-luciferaseconstruct (androgen responsive), and an SV40-renilla luciferaseconstruct as an internal control. The following day, the cells weresplit and drugs added in the presence of 3 nM DHT. Luciferase productionwas measured the following day (Dual luciferase assay kit, Promega).

No compounds had androgen activity in the absence of DHT in either theconformation or the transcription reporter assays, even at dosesexceeding 10 uM. PP and HH were much more effective than the competitiveantagonists OH—F and bicalutamide (BiC) at inhibiting reportertranscription (Table 1), so the anti-androgen characteristics of thesecompounds were studied further. Both PP and HH inhibited DHT-responsivegene expression with efficacies far superior to BiC (Table 1). To ruleout the possibility that the pamoate salt of pyrvinium was mediatingtranscriptional inhibition, anion exchange (Dowex) was used to replacethe pamoate with a chloride ion. Pyrvinium chloride was just aseffective as pyrvinium pamoate (FIG. 4).

TABLE 1 IC₅₀ of various compounds identified in screens for novelanti-androgens. Activity on PSA-luciferase Activity on PSA-luciferaseIC₅₀(nM) IC₅₀(nM) Compound LNCaP LAPC4 PP 24 12 HH 127 106 OH—F agonist130 Casodex 585 1,571 Esculin 62,159 831 Scopoletin 603,586 1,378Warfarin 4,500 1,954 Harmine 1,560 80 Harmaline 1,190 nd Harman 380 ndNorharman 2,500 nd PP = pyrvinium pamoate; HH = harmol hydrochloride;OH—F = hydroxy-flutamide; BiC = bicalutamide. nd = no data.

Example 3 Assay for Inhibition of an Androgen Receptor

The assay below demonstrates that the compounds identified in Example 2inhibit endogenous androgen receptor gene expression and are notcompetitive antagonists.

To confirm that the effects of the lead compounds hold true forendogenous AR-regulated gene expression, quantitative RT-PCR (qRT-PCR)was used to monitor effects on a select number of androgen-responsivegenes in LAPC4 and LNCaP cells. LAPC4 and LNCaP cells were grown in thepresence or absence of 3 nM DHT in charcoal-stripped media, with orwithout the inhibitors. RNA was harvested using an RNAeasy kit (Qiagen)and reverse transcribed (MMLV-RT, Invitrogen). Real-time PCR was carriedout on a 7300 Real Time PCR System (Applied Biosystems) using SYBR greenas the detecting dye and Rox as the reference dye. The androgenresponsive genes kalikrein 3, or PSA (KLK3) [Cleutjens, K. B., et al.,Two androgen response regions cooperate in steroid hormone regulatedactivity of the prostate-specific antigen promoter. J Biol Chem, 1996.271(11): p. 6379-88; Nelson, P. S., et al., The program ofandrogen-responsive genes in neoplastic prostate epithelium. Proc NatlAcad Sci USA, 2002. 99(18): p. 11890-5), metalloproteinase 16 (MMP16)(Nelson, P. S., et al., The program of androgen-responsive genes inneoplastic prostate epithelium. Proc Natl Acad Sci USA, 2002. 99(18): p.11890-5), transmembrane protease serine 2 (TMPRSS2) (Nelson, P. S., etal., The program of androgen-responsive genes in neoplastic prostateepithelium. Proc Natl Acad Sci USA, 2002. 99(18): p. 11890-5; Aronin,N., et al., Are there multiple pathways in the pathogenesis ofHuntington's disease? Philosophical Transactions of the Royal Society ofLondon. Series B: Biological Sciences, 1999. 354(1386): p. 995-1003;Vaarala, M. H., et al., Expression of transmembrane serine proteaseTMPRSS2 in mouse and human tissues. J Pathol, 2001. 193(1): p. 134-40),FK506-binding immunophilin 51 (FKBP51) (Nelson, P. S., et al., Theprogram of androgen-responsive genes in neoplastic prostate epithelium.Proc Natl Acad Sci USA, 2002. 99(18): p. 11890-5; Amler, L. C., et al.,Dysregulated expression of androgen-responsive and nonresponsive genesin the androgen-independent prostate cancer xenograft model CWR22-R1.Cancer Res, 2000. 60(21): p. 6134-41; Velasco, A. M., et al.,Identification and validation of novel androgen-regulated genes inprostate cancer. Endocrinology, 2004. 145(8): p. 3913-24; Magee, J. A.,et al., Direct, androgen receptor-mediated regulation of the FKBP5 genevia a distal enhancer element. Endocrinology, 2006. 147(1): p. 590-8),G-protein coupled receptor RDC1 homolog, or chemokine orphan receptor 1(RDC-1) (Nelson, P. S., et al., The program of androgen-responsive genesin neoplastic prostate epithelium. Proc Natl Acad Sci USA, 2002. 99(18):p. 11890-5), NK homeobox family member 3 (NRx3.1) (Nelson, P. S., etal., The program of androgen-responsive genes in neoplastic prostateepithelium. Proc Natl Acad Sci USA, 2002. 99(18): p. 11890-5; Bieberich,C. J., et al., Prostate-specific and androgen-dependent expression of anovel homeobox gene. J Biol Chem, 1996. 271(50): p. 31779-82;Aboody-Guterman, K. S., et al., Green fluorescent protein as a reporterfor retrovirus and helper virus-free HSV-1 amplicon vector-mediated genetransfer into neural cells in culture and in vivo. Neuroreport, 1997.8(17): p. 3801-8) were all normalized to the transcription of thehousekeeping ribosomal gene (RPL19). All samples were done in triplicateand separately normalized. KLK3, NRx3.1, TMPRSS2, and FKBP51 were allinduced by treatment with DHT, and this induction was inhibited tovarying degrees by BiC, PP, and HH. Likewise, MMP-16 and RDC-1 wererepressed by treatment with DHT, and the repression was lifted tovarying degrees by all of the AR inhibitors. Table 2 summarizes theresults from 3 or 4 separate qRT-PCR experiments in each cell type. Twogenes known to be induced by DHT in LNCaPs, NRx3.1 and TMPRSS2, were notsignificantly induced in LAPC4 cells. Both PP and HH were observed toreadily suppress expression of several androgen-responsive genes atleast as effectively as BiC (Table 2).

TABLE 2 PP and HH each inhibit gene expression mediated by endogenousAR. Both PP and HH reduced androgen-induced gene induction in a mannercomparable or superior to BiC. Evaluation of androgen-repressed genes(shown in the bottom section) indicated that the inhibitors eachde-repressed expression. No 3 nM 3 nM DHT/ 3 nM DHT/ 3 nM DHT/ DHT DHT 1μM BIC 100 nM PP 100 nM HH LNCaP transcription (normalized to RPL19)KLK3 1 1.89 1.53 1.57 1.46 Nkx3.1 1 2.00 1.31 0.78 1.28 FKBP51 1 2.153.56 1.46 1.90 TMPRSS2 1 3.70 2.58 0.92 1.22 MMP16 1 0.61 0.78 1.57 1.02RDC1 1 0.66 0.66 0.94 1.22 LAPC4 transcription (normalized to RPL19)KLK3 1 1.97 1.70 1.61 1.64 Nkx3.1 1 1.08 1.11 0.77 1.55 FKBP51 1 21.421.32 9.55 11.96 TMPRSS2 1 1.13 1.23 1.90 1.86 MMP16 1 0.71 2.32 2.924.74 RDC1 1 0.63 2.07 1.22 2.88

PP and HH are Non-Competitive Antagonists.

As a first step to determine whether PP and HH function as competitiveor noncompetitive antagonists of AR, DHT was titrated in LNCaP cellstransiently transfected with a PSA-luciferase reporter, and treated witha moderate dose of each inhibitor. DHT overcame the inhibitory effect ofOH—F, producing maximal activation. By contrast, both PP and HHinhibited maximal DHT-induced activation, despite very high final DHTconcentrations (FIG. 5). This was consistent with activity asnon-competitive antagonists.

Example 4 Synergistic Behavior of PP, HH and BiC

The presence of synergy in the use of PP, HH and BiC as androgenreceptor antagonists is confirmed using the procedure below.

The existence of synergy between the lead compounds and BiC was testedfor using LAPC4 and LNCaP cells. When added in various combinations toLNCaP and LAPC4 cells transfected with the PSA-luciferase, both PP andHH synergized with each other, and with BiC (Table 3). Synergism can bedefined as a combination index of less than one, using the non-exclusiveassumption described by Chou et al. (Chou, T. C. and P. Talalay,Quantitative analysis of dose-effect relationships: the combined effectsof multiple drugs or enzyme inhibitors. Adv Enzyme Regul, 1984. 22: p.27-55). The synergy between PP, HH, and BiC is indicates that theyfunction by distinct mechanisms.

TABLE 3 Synergy between PP, HH and BiC. Dose response curves werecarried out with the inhibitors in the indicated ratios, using 3 nM DHTto activate transcription from PSA-Luciferase in the indicated celltypes (LNCaP or LAPC4). The expected IC50 value is reported for thefirst drug indicated in the combination. The Combination Index is acalculated measure of “synergy” based on Chou et al. (1984); numberslower than one indicate synergy. Expected Combination Combination CellType IC₅₀ (nM) Actual IC₅₀ (nM) Index at f50 PP:HH 1:1 LNCaP 9.27 8.270.54 PP:HH 1:1 LAPC4 7.83 4.57 0.28 PP:BIC 1:1 LNCaP 18.47 12.23 0.33PP:BIC 1:1 LAPC4 18.25 0.64 0.02 HH:BIC 1:1 LNCaP 78.05 31.85 0.21HH:BIC 1:1 LAPC4 142.1 28.33 0.1 PP:HH 1:10 LNCaP 26.79 2.56 0.08 PP:BIC1:10 LNCaP 27.21 0.4 0.01 PP:BIC 1:30 LNCaP 27.69 10.25 0.44 PP:BIC1:100 LNCaP 27.09 2.38 0.32 PP:OHF 1:1 LAPC4 7.56 5.79 0.39 Harmol:OHFLAPC4 93.06 66.18 0.38

Example 5 Growth Inhibition by PP and HH

The procedures set-forth below were used to determine whether PP and HHwould affect the androgen-dependent proliferation of LAPC4 and LNCaPcells in cell culture, as well as the androgen-independent growth ofLN-AR cells that over-express AR, and which are a model of “androgenindependent” prostate cancer.

LN-AR cells were created by retroviral infection of a high-expressing ARvector into LNCaP cells, and proliferate independent of androgen in cellculture, unlike their parental line (Chen, C. D., et al., Moleculardeterminants of resistance to antiandrogen therapy. Nat Med, 2004.10(1): p. 33-9). HEK293 cells were used as a control. Cells weretransferred to charcoal-stripped media two days before they were splitand plated in quadruplicate at a density of approximately 20,000cells/well in 48-well plates. The following day, medium with or without3 nM DHT was added to the cells with or without PP (100 nM), HH (100nM), or BiC (1 μM). Media were changed every day, using a singlepreparation to ensure consistent compound concentrations. Proliferationwas determined by measuring the DNA content of the cells in each well.Each day, the cells in four wells were fixed in 100% cold methanol,followed by staining for 5 min at RT with 0.2 ng/mL4′,6-diamidino-2-phenylindole (DAPI) in PBS. The cells were washed oncein PBS, then read on a fluorescence plate reader using 365/439excitation/emission wavelengths. LAPC4 and LNCaP cells proliferated to amuch greater degree in the presence of DHT, while LN-AR cellsproliferated in the presence and absence of DHT. PP was the onlycompound able to inhibit the growth of all four cell lines after sevendays, while HH and BiC only inhibited significantly the growth of LNCaPcells. Control HEK293 cells were not significantly affected by any ofthe compounds, suggesting that none of them has non-specificgrowth-inhibitory effects. PP and HH worked at least as well as BiC toblock androgen-induced proliferation (FIG. 6).

Example 6 Pharmacokinetics and Toxicity of Compounds in Mice

Anti-androgen activity of PP and HH in mice were determined using theprocedure set forth below.

Wild-type FVB mice were given a single intraperitoneal injection ofeither PP or HH. Serum samples were obtained at fixed time intervals,and the drug levels determined by mass spectrometry. These resultsindicated that HH was rapidly cleared. PP exhibited a prolongedhalf-life indicating that it could be a suitable therapy (Table 4).Toxicity of PP was tested using various doses ranging from 0.1 to 10mg/kg. 10 mg/kg was toxic, while the mice tolerated 5 mg/kg.

TABLE 4 Pharmacokinetics of PP vs. HH. Mice were given a single IPinjection or PO dose of PP or HH at 5 mg/kg. Serum samples were drawn atthe indicated time points, and blood levels measured using massspectrometry. 0.25 hr 1 hr 6 hr 24 hr Plasma [PP] (ng/mL) PO 40.2 13.5nd nd IP 57.2 19.7 15.9 6.9 Plasma [HH] (ng/mL) PO  6.7 nd nd nd IP 32.3 5.0 nd nd

PP Synergizes with BiC In Vivo to Cause Prostate Atrophy.

As a positive control, one cohort was castrated four weeks prior totissue recovery, one cohort was untreated, and the other cohorts weretreated either with 1 mg/kg PP, 100 mg/kg BiC, or PP/bicalutamide (BiC)in combination. Animals were treated 5 times per week for four weeks. Atthe end of the study period, animals were sacrificed and prostate tissueweighed. After weighing, the tissue was divided in half for pathologicaland genetic studies. BiC treatment decreased prostate tissue size by 35%(p<0.003). Treatment with PP:BiC caused a further reduction by 63% thatwas highly significant vs. the effect of BiC alone (P<0.0005). PP alonedid not produce a statistically significant effect. (FIG. 7).

PP Synergizes with BiC In Vivo to Inhibit Androgen-Dependent ProstateGene Expression.

To evaluate the effect of PP and PP:BiC on androgen-dependent geneexpression in the prostate, total RNA was prepared from half of theprostate glands isolated in the in vivo trial. RT-PCR was carried out,and gene expression was determined relative to RPL19, an endogenousprostate gene that is completely androgen unresponsive (E. Bolton, K. R.Yamamoto: unpublished data). Five androgen-responsive genes that areexpressed throughout all lobes of the mouse prostate were evaluated. Ineach case, it was observed that PP and BiC reduced gene expressionsignificantly, but their combination was superior. In most cases, thecombination approached that of castrated animals (FIG. 8). Takentogether with the preceding experiment, PP exerts anti-androgen effects,and synergy with BiC as an anti-androgen in vivo.

Example 7 Effects of PP, THP, and BiC on the Mouse Prostate

10 FVB male mice (12 wk) were treated with vehicle, 1 mg/kg pyrviniumpamoate (PP), 100 mg/kg bicalutamide (BiC), 2 mg/kg THP, 20 mg/kg, or acombination of 2 mg/kg THP and 100 mg/kg BiC for 5 weeks, daily, M-Fdosing. A cohort of mice were castrated as a positive control.

Treatment with BiC, PP, THP+BiC, or castration caused a significant (byanalysis of variance) decrease in prostate wet weight. 20 mg/kg THPcaused an abnormal milky appearance of the prostate, though it did notreduce overall wet weight (FIG. 11)

Mice treated with THP or BiC alone had modest atrophy in the dorsalprostate (by histological examination) as well as other lobes, whereas acombination of the two caused severe atrophy similar to that caused bycastration (FIG. 12).

For quantitative PCR, RNA was isolated from mouse prostates andreverse-transcribed. The expression of several androgen-regulated genes,of which a subset is shown (FIG. 13), was determined by using QPCR andnormalizing to the expression of the housekeeping gene RPL19. While 2mg/kg treatment of THP had modest effects, 20 mg/kg THP treatmentsignificantly decreased the transcription of many androgen regulatedgenes in the prostate.

Example 8 Efficacy of THP, THP:BiC, and SHP Against AR Transcription inLAPC4 Cells

LAPC4 cells, a prostate cancer cell line that expresses native androgenreceptor, were transfected with a PSA-luciferase reporter, and aCMV-renilla luciferase control. Cells were treated with DHT andincreasing amounts of tetrahydropyrvinium (THP), as indicated in FIG.14. Normalized luciferase activity was determined, which demonstratesstrong inhibition of androgen receptor-mediated transcription bytetrahydropyrvinium.

In a separate experiment, cells were treated with DHT or a titration ofa 1:30 tetrahydropyrvinium/bicalutamide combination to test for synergy.The combination of the two drugs exceeded the transcriptional inhibitionpredicted by an additive effect, indicating synergistic inhibition ofthe androgen receptor (FIG. 15).

In a second separate experiment, the activity of SHP and THP againstPSA-luciferase AR-responsive promoter was determined (FIG. 16).

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. In addition, each reference provided herein isincorporated by reference in its entirety to the same extent as if eachreference was individually incorporated by reference.

What is claimed is:
 1. A method of inhibiting the androgen receptor, themethod comprising: administering to a patient in need thereof, atherapeutically effective amount of a compound of Formula I:

wherein each R¹ is independently selected from the group consisting ofhydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ alkoxy, —SR⁴,—NR⁴R⁵, cycloalkyl, heterocycloalkyl, aryl and heteroaryl; R² isselected from the group consisting of hydrogen, C₁₋₆ alkyl, C₁₋₆alkyl-OH, C₂₋₆ alkenyl and C₂₋₆ alkynyl; R³ is aryl, optionallysubstituted with from 1 to 3 R⁶ groups; R⁴ and R⁵ are each independentlyselected from the group consisting of hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyland C₂₋₆ alkynyl; or R⁴ and R⁵ are combined with the nitrogen to whichthey are attached to form a heterocyclic ring having from 5 to 7 ringmembers and from 1 to 3 heteroatoms each independently selected from thegroup consisting of N, O and S; each R⁶ is independently selected fromthe group consisting of H, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl andC₁₋₆ alkoxy; L is a linker selected from the group consisting of C₁₋₆alkylene, C₂₋₆ alkenylene, C₂₋₆ alkynylene and C₃₋₆ cycloalkylene; X isa heteroaryl ring having from 5 to 6 ring members and from 1 to 3heteroatoms each independently selected from the group consisting of N,O and S, optionally substituted with from 1 to 3 R⁸ groups; each R⁸ isindependently selected from the group consisting of H, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl and C₁₋₆ alkoxy; and salts, hydrates and prodrugsthereof, thereby inhibiting the androgen receptor.
 2. The method ofclaim 1, wherein the compound is selected from the group consisting of


3. The method of claim 1, wherein the salt forms comprise a counterionselected from the group consisting of pamoate, chloride, bromide,succinate, maleate and acetate.
 4. The method of claim 1, wherein themethod of inhibiting treats a disease selected from the group consistingof prostate cancer, ovarian cancer, hepatocellular carcinoma, acnevulgaris, endometriosis, acanthosis nigricans, hypertrichosis, breastcancer, precocious puberty, polycystic ovary syndrome, benign prostatichyperplasia, alopecia, hirsutism and hypersexuality/paraphilia.
 5. Themethod of claim 4, wherein the disease is prostate cancer.
 6. The methodof claim 5, wherein the disease is primary prostate cancer.
 7. Themethod of claim 5, wherein the disease is hormone refractory prostatecancer.
 8. The method of claim 4, wherein the administration is viatopical, oral, intravenous, intradermal, intramuscular or parenteraladministration.
 9. The method of claim 4, wherein the disease isalopecia and the administration is topical.
 10. The method of claim 1,wherein the compound of Formula I is administered with a course ofhormonal therapy, wherein the compound for hormonal therapy is selectedfrom the group consisting of an anti-androgen and a LnRH agonist. 11.The method of claim 10, wherein the compounds are administeredseparately.
 12. The method of claim 10, wherein the compounds areadmixed.
 13. The method of claim 10, wherein the compounds areadministered at the same time.
 14. The method of claim 10, wherein thecompounds are administered at different times.
 15. The method of claim10, wherein the compound of Formula I is administered in combinationwith a therapeutically effective amount of a compound selected from thegroup consisting of docetaxel, paclitaxel, bicalutamide, flutamide,hydroxyflutamide, nilutamide, spionolactone, cyproterone acetate,ketoconazole, finasteride and dutasteride.
 16. The method of claim 10,wherein the compound of Formula I is administered in combination with atherapeutically effective amount of a coumarin.
 17. The method of claim10, wherein the compound of Formula I is administered in combinationwith a therapeutically effective amount of bicalutamide.